ExKine Total Protein Extraction Kit

Transcription

1 ExKine Total Protein Extraction Kit Item NO. KTP3006 Product Name ExKine Total Protein Extraction Kit ATTENTION For laboratory research use only. Not for clinical or diagnostic use Version

2 TABLE OF CONTENTS INTRODUCTION Background & Principle... 1 Storage/Stability... 1 Assay restrictions... 1 PRODUCT INFORMATION Materials supplied and Storage conditions... 2 Other supplies required, Not Supplied... 2 Precautions... 2 Technical hints... 2 ASSAY PROTOCOL Reagent Preparation... 3 Recommended procedures... 3 Version

3 INTRODUCTION Background & Principle Extraction of cellular proteins requires efficient cell lysis and protein solubilization, while avoiding protein degradation and/or interference with protein immunoreactivity and biological activity. The Total Protein Extraction Kit is composed of rapid and efficient solutions for lysis of mammalian adherent cells, nonadherent cells, and tissues, which is compatible with many different applications, such as reporter assays, protein purification, Western blot, immunoprecipitation. Storage/Stability Stable for at least 12 months at -20 C from date of shipment. Gel pack with blue ice. Assay Restrictions Assay kit is intended for research use only. Not for use in diagnostic procedures. Do not mix or substitute reagents or materials from other kit lots or vendors. Kits are QC tested as a set of components and performance cannot be guaranteed if utilized separately or substituted. 1

4 PRODUCT INFORMATION Materials supplied and Storage conditions Kit components Quantity 50T 200T Storage conditions Protein Extraction Reagent (2 ) 25 ml 100 ml -20 C Protease Inhibitor (100 ) 0.5 ml 2 ml -20 C Other supplies required, Not Supplied Microcentrifuge Pipettes and pipette tips Phosphate-buffered saline (PBS) Glass tissue homogenizer Cell scrapers Technical hints To avoid cross-contamination, change pipette tips between sample additions, and between reagent additions. Also, use separate reservoirs for each reagent. Ensure all reagents and solutions are at the appropriate temperature before starting the assay. Make sure all necessary equipment is switched on and set at the appropriate temperature. 2

5 ASSAY PROTOCOL Reagent Preparation Protein Extraction Reagent (2 ): Keep on ice while using. Dilute to a 1 solution using ddh 2O. Immediately before use, add 10 µl protease inhibitors to each of 1 ml of Protein Extraction Reagent (1 ). Note: 1. If the buffer will be continually used, it is recommended that the 5 buffer be kept at 4 C for 1-2 weeks. For longer storage, it is recommended aliquot and stored at 20 C. 2. If desired, add phosphatase inhibitors to the buffer immediately before use. Recommended procedures Note: Perform all steps at 2 8 C. Use precooled buffers and equipment. Ensure all the solutions are defrosted and homogeneous. A. For adherent cells 1. Remove the growth media from the cells and wash the cells twice with PBS. 2. Add 1 ml of 1 Protein Extraction Reagent to 10 cm dish. Note: Cells grown in 10cm plates typically contain 10 7 cells (50mg). 3. Incubate the plate on ice for 5 minutes, swirling occasionally for uniform spreading. 4. Scrape and collect the lysate to a microcentrifuge tube. Note: To increase the protein yields, sonicate the lysate briefly. 5. Centrifuge samples at 14,000 g for 15 minutes to pellet the cell debris and transfer the supernatant to a new tube for further analysis. B. For non-adherent cells 1. Pellet the cells by centrifugation at 700 g for 10 minutes. 2. Discard the supernatant and wash with ice-cold PBS twice. 3. Add 1 ml of 1 Protein Extraction Reagent to cells. Pipette the mixture up and down to suspend the pellet. 4. Shake mixture gently for 15 minutes on ice. To increase the protein yields, sonicate the lysate briefly. 5. Centrifuge samples at 14,000 g for 15 minutes to pellet the cell debris and transfer the supernatant to a new tube for further analysis. 3

6 C. For Tissue Preparation 1. Cut mg of tissue into small pieces and place in a microcentrifuge tube. 2. Wash tissue with PBS. Centrifuge tissue at 500 g for 5 minutes and discard the PBS. 3. Resuspend the tissue gently in 1mL ice-cold 1 Protein Extraction Reagent. 4. Homogenize tissue using a Dounce homogenizer or a tissue grinder until the cells are completely lysed. 5. Centrifuge samples at 14,000 g for 15 minutes to pellet the cell debris and transfer the supernatant to a new tube for further analysis. 4